The zonula occludens toxin (ZOT) of Vibrio cholerae is a newly recognized toxin which disrupts the intercellular tight junctions of rabbit intestinal epithelium. ZOT is and important toxin to study because it has been correlated with the production of diarrhea. The long term objective of this project will be to determine the importance of ZOT in V. cholerae pathogenesis. This project will study three distinct aspects of ZOT. 1) Regulation of zot -The zot promoter will first be mapped by primer extention. Next V. cholerae will be subjected to variations in temperature, salt, Ph, and iron to determine if any conditions increase or decrease expression of zot. Also, a possible decrease in expression of zot by ToxR will be analyzed. 2) Active site - N- and C-terminal and internal deletions of zot will be created an analyzed for loss of activity. Once a functional area is identified, site-directed mutagenesis will be used to identify important functional residues. 3) Mechanism of action - In order to determine how the tight junctions are being disrupted, the effects of ZOT on signal transduction pathways, microfilament organization, and molecules of the tight junctions will be studied.